Filamentous fungal associates of the alder bark beetle, Alniphagus aspericollis, including an undescribed species of Neonectria

Bark beetles (Coleoptera: Curculionidae; Scolytinae) are tree-infesting insects that consume subcortical tissues and fungi. Species capable of killing their host trees are most commonly associated with conifers, as very few bark beetle species infest and kill hardwood hosts directly. The alder bark beetle, Alniphagus aspericollis, is a hardwood-killing bark beetle that colonizes and kills red alder, Alnus rubra. Conifer-killing bark beetles have well-known associations with symbiotic ophiostomatoid fungi that facilitate their life histories, but it is unknown whether A. aspericollis has any fungal associates. This study was conducted to identify any consistent filamentous fungal associates of A. aspericollis and characterize the consistency of observed beetle–fungus relationships. Beetles and gallery phloem samples were collected from seven sites throughout the Greater Vancouver region in British Columbia, Canada. Filamentous fungi were isolated from these samples and identified by DNA barcoding using the internal transcribed spacer (ITS) region and other barcode regions for resolution to the species-level for the most dominant isolates. The most common fungal associate was a previously undescribed Neonectria major-like fungus, Neonectria sp. nov., which was isolated from ~67% of adult beetles, ~59% of phloem samples, and ~94% of the beetle-infested trees. Ophiostoma quercus was isolated from ~28% of adult beetles, ~9% of phloem samples, and ~56% of infested trees and deemed a casual associate of A. aspericollis, while a putatively novel species of Ophiostoma was more infrequently isolated from A. aspericollis and its galleries. Cadophora spadicis, a new record for red alder, was rarely isolated and is probably coincidentally carried by A. aspericollis. Overall, A. aspericollis was only loosely associated with ophiostomatoid fungi, suggesting that these fungi have little ecological significance in the beetle–tree interaction, while Neonectria sp. nov. may be a symbiote of A. aspericollis that is vectored by the beetle.

Introduction Symbiotic fungal partners of A. aspericollis, if any, are yet to be discovered. Borden [36] observed that infestation by A. aspericollis could precede wood rot but did not suggest that fungal inoculation was directly or indirectly facilitated by A. aspericollis. Previous studies of the fungal communities of red alder in the Pacific Northwest have recorded various species of saprotrophic decay fungi and non-ophiostomatoid pathogens but no associations with A. aspericollis were reported [39,40].
This study explores the filamentous fungal community associated with A. aspericollis. Our objectives were to determine if A. aspericollis is associated with fungi and characterize the consistency of any beetle-fungus relationships. Given the ability of A. rubra to fix nitrogen from the atmosphere via symbiotic root nodule actinomycetes [37,38], we also tested the prediction that A. aspericollis would not require, and therefore not be consistently associated with, nitrogen-provisioning ophiostomatoid fungi in the same manner as conifer-feeding bark beetles that depend on these fungi to complete their development [21][22][23].

Beetle and gallery phloem collection
Adult A. aspericollis (Fig 1) were collected from seven sites across the Greater Vancouver region in British Columbia, Canada between 14 July and 26 August 2015 (Fig 2). Research permits were obtained from Metro Vancouver Regional Parks (Fig 2 sites 2-4, 6), the District of West Vancouver (site 1), the City of Port Coquitlam (site 5), and the City of Burnaby (site 7). One to three infested red alder hosts were located at each site, and three adult beetles were obtained from under the bark of each tree (Figs 1 and 2). A total of 54 individual beetles were extracted from separate entrance holes and non-overlapping galleries. Larvae, pupae and teneral adults were also collected when present. All specimens were individually transferred into separate vials with ethanol-sterilized tweezers. A section of phloem (approximately 5 x 5 cm) was also extracted from each of the sampled beetle galleries (n = 54) using a hammer and chisel. All beetle and gallery phloem samples were transported back to the laboratory and refrigerated at 4˚C until fungal isolations were conducted.

Fungal isolations
Fungal isolations from the beetle and phloem samples were performed within one week of collection. Each beetle was thoroughly washed with 100 μl of sterile water and 50 μl of the resulting liquid inoculum was thinly spread onto malt extract agar (MEA) in a petri dish. From each phloem sample, five thinly-sliced chips (approximately 1 x 1 cm) were sectioned out with sterilized instruments, particularly around the entrance hole and gallery regions. Phloem chips were rapidly surface sterilized using 70% ethanol, rinsed thoroughly with sterile water, and plated onto water agar in a petri dish for incubation at room temperature.
Individual filamentous fungal colonies emerged in the initial isolation dishes within three days of incubation. Since the colonies in each dish were generally too numerous to be individually isolated, they were first sorted into different morphotypes based upon visible characteristics (e.g., colony diameter, texture, hyphal/spore colour, margin characteristics). Representative samples and variants of each morphotype were then sub-cultured onto MEA to ensure that maximum fungal diversity was captured per initial isolation dish. Sub-cultures were incubated at room temperature, and repeatedly sub-cultured until pure isolates were obtained. Numerous yeast and bacterial colonies were also observed in most of the initial isolation dishes but were not sub-cultured as these taxa were beyond the scope of this study. rDNA gene region, which is a universal DNA barcode for all fungal species [41], was amplified from all samples using the primers ITS1F and ITS4 [42,43]. PCR was performed with the ABI 2720 Thermal Cycler (Applied Biosystems, Foster City, California, USA) or the T100 TM Thermal Cycler (Bio-Rad, Hercules, California, USA) using a 27 μl reaction system and the following thermal cycler program: 94˚C for 3 min, followed by 35 cycles of 94˚C for 30 s, 55˚C for 30 s, 72˚C for 1 min, and then held for 10 min at 72˚C. PCR amplicons were purified using Unifilter TM GF/C 384-well 100 μl microplates (Whatman Inc., Ann Arbor, Michigan, USA) and Sanger sequenced with the ABI 3730xl DNA Sequencer (Applied Biosystems, Foster City, California, USA) at the Centre de Recherche du CHUL (CHUQ), Université Laval (Quebec, Canada). ITS1F was used as the forward sequencing primer to produce single-strand sequences for all amplicons.

Sequence analysis and fungal identification
Raw sequences were edited and verified using BioEdit Sequence Alignment Editor software [51] version 7.1 (2011). Resulting ITS sequences were compared to existing GenBank sequence data using a BLAST similarity search (BLASTn) to identify each isolate to the genus-or species-level. To verify the species identities of the most frequently isolated morphotypes, singlelocus phylogenetic trees were constructed via maximum likelihood analysis, using PhyML 3.0 [52] with the HKY85 substitution model. Phylogenies were constructed for each Neonectria, Ophiostoma, and Cadophora gene region. The species identity of each frequently-isolated morphotype was determined based on its phylogenetic placement relative to other closely-related species. Comparative phylogenetic data were derived from the most current relevant studies that also provided appropriate outgroups for tree rooting. For Neonectria isolates, N. castaneicola was chosen as the outgroup for the ITS dataset [45], while the more related N. fuckeliana was selected as the outgroup for the RPB2 dataset [47].

Phylogenetic analyses and species identification
Isolates from the genus Neonectria (strains CGL 401 and CGL 402) were identified as an undescribed N. major-like species, hereafter Neonectria sp. nov., based upon morphological culture characteristics, existing host-specificity data, and by comparison of ITS, RPB2, and EF1-α sequences with those of closely-related Neonectria species (Fig 3) [45,47,56]. Overall, our phylogenetic analyses placed these Neonectria isolates most closely to N. major and N. ditissima but within a well-supported distinct clade (88-98% bootstrap support). DNA amplification of Neonectria sp. nov. isolates resulted in fragments of~500 bp for the ITS barcode region,~960 bp for the partial RPB2 gene region, and~260 bp for the partial EF1-α gene region. The identity of Neonectria sp. nov. could not be fully resolved by phylogenetic analysis of the ITS barcode region, which grouped the isolates with species such as N. major, N. ditissima, and N. neomacrospora ( Fig 3A). Phylogenetic analysis of the partial RPB2 gene region placed the Neonectria sp. nov. isolates within a single well-supported clade that was most closely related to N. major and N. ditissima, thereby distinguishing the species from N. neomacrospora and N. ditissimopsis ( Fig 3B). Analysis of the partial EF1-α gene region also placed the isolates within a single well-supported clade that was related to, but distinct from, N. ditissimopsis and slightly more distantly related to N. major and N. ditissima ( Fig 3C). Neonectria major, but not N. ditissima, has been isolated from Alnus hosts and the cultural characteristics of our Neonectria sp. nov. isolates (e.g., regularly-tufted aerial mycelia, saffron pigmentation on underside) ( Fig 4A) are in agreement with existing descriptions of N. major [47], leading us to conclude that the Neonectria sp. nov. isolates observed during this study are N. major-like. Isolates from the genus Ophiostoma were identified to the species-level based upon comparison of ITS, BT and EF1-α sequences with those of closely-related species [57][58][59][60][61][62][63] in the "O. ulmi complex" [64]. DNA amplification of Ophiostoma isolates resulted in fragments of~600 bp for the ITS barcode region,~400 bp for the partial BT gene region, and~460-540 bp for the partial EF1-α gene region. Phylogenetic analyses conducted for all three gene regions split the Ophiostoma isolates into two distinct groups (Fig 5). a "Neonectria galligena" is now synonymous with N. ditissima, with the latter recognised as the official name [47].
e Placed into the genus Cosmospora by [54].
f From the phylogenetic study of Cosmospora by [55].
https://doi.org/10.1371/journal.pone.0284393.t001 Cladosporium sp. Unknown Ascomycota spp. The first group of Ophiostoma isolates (strains CGL101 to CGL113) was identified as O. quercus by phylogenetic analysis. The identity of this group could not be fully resolved to the species-level by analysis of the ITS barcode region, as it did not resolve separately from species from the "O. ulmi complex" (Fig 5A). Analysis of the partial BT gene region placed these isolates into two clades that grouped together with O. quercus isolates to form a single well-supported clade (Fig 5B). Similarly, analysis of the partial EF1-α gene region grouped all isolates with O. quercus to form a single well-supported clade (Fig 5C).
The second group of Ophiostoma isolates (strain CGL201) was identified by phylogenetic analysis as a putatively novel Ophiostoma sp. During analysis of the ITS barcode region, this group formed a single well-supported clade that was related to, but distinct from, O. borealis, O. bacillisporum, and O. catonianum (Fig 5A). Similarly, analysis of the partial BT gene region placed this group in a distinct and well-supported clade that was related to, but separate from, O. catonianum (Fig 5B). Phylogenetic analysis of the partial EF1-α gene region also placed this group in a single well-supported clade that was related to, but distinct from, O. karelicum (Fig 5C). The consistent, well-supported distinction of this group from other species in the "O. ulmi complex" strongly suggests the identification of an Ophiostoma species that has yet to be characterized.
Isolates from the genus Cadophora (strains CGL301 to CGL303) were identified as C. spadicis, based on comparison of ITS, EF1-α and BT sequences with those of closely-related Cadophora species [50] (Fig 6). DNA amplification of Cadophora isolates resulted in fragments of 500 bp for the ITS barcode region and~500 bp for the partial EF1-α gene region. Phylogenetic analysis of the ITS barcode region grouped all isolates with C. spadicis to form a single well-supported clade (Fig 6A). Analyses of the partial EF1-α gene region and the partial BT gene region for Cadophora isolates produced similar results to align with C. spadicis (Fig 6B, 6C).
The sole isolate from the ophiostomatoid genus Graphilbum was identified as G. fragrans based upon the closest BLAST matches of its ITS barcode sequence (Table 1) and culture morphology (e.g., production of yellow pigmentation in MEA, as described by [65,66]) (Fig 4E). Single isolates of Valsalnicola oxystoma and Chondrostereum purpureum were also identified to the species-level by BLAST searches of their ITS barcode sequences ( Table 1). Given that G. fragrans, V. oxystoma and C. purpureum were each only represented by one isolate, and no comprehensive genera-level phylogenetic studies are currently available for these species, no further phylogenetic analyses were conducted for these isolates.
Neonectria sp. nov. was the most frequently isolated filamentous fungus in our study, associated with approximately two-thirds of adult beetles and gallery phloem samples and almost all beetle-infested trees ( Table 2). This canker fungus was concurrently isolated from both adult beetles and phloem samples collected from more than half of the sampled galleries. Aside from Pacific Spirit Regional Park, and Tynehead Regional Park in the case of phloem isolations, Neonectria sp. nov. was isolated from more than 50% of adult beetles and gallery phloem samples collected at each study site (Table 3). Strikingly, Neonectria sp. nov. was isolated from every beetle-infested tree at each site except Pacific Spirit Regional Park, where two out of the three sampled trees yielded Neonectria sp. nov. isolates. At each site, however, isolation of Neonectria sp. nov. never exceeded 90% for either adult beetle isolations or gallery phloem samples. Neonectria sp. nov. was only occasionally isolated from A. aspericollis larvae, pupae, and teneral adults.
Ophiostoma quercus was the second most frequently isolated filamentous fungus, but its association with A. aspericollis was much less consistent than Neonectria sp. nov. Additionally, O. quercus was concurrently isolated from adult beetles and phloem samples from only 5.6% of sampled galleries (Table 2), nearly ten times lower than that for Neonectria sp. nov. At five of seven study sites, O. quercus was consistently isolated from 33% of adult beetles, with the exceptions of Pacific Spirit Regional Park and Cypress Mountain where isolation rates were 56% and 11%, respectively (Table 3). Isolation frequency of O. quercus was even lower for gallery phloem samples from each site, with three sites yielding no isolates of O. quercus from phloem at all. Beetle-infested trees surveyed at five of seven sites produced no isolates of O. quercus, and at Aldergrove Regional Park, O. quercus was not isolated from any beetle or gallery phloem sample. Ophiostoma quercus was almost never isolated from larvae, pupae, or teneral adults, the only exception being a single larva-derived isolate from Tynehead Regional Park.
Ophiostoma sp. nov. was rarely isolated, with an overall occurrence of just 3.7-16.7% on adult beetles, in galleries, and infested trees ( Table 2). This was primarily because Ophiostoma sp. nov. was only isolated from Burnaby Mountain, where 33% of adult beetles and 22% of gallery phloem samples yielded the fungus (Table 3). Ophiostoma sp. nov. was isolated from all 3 beetle-infested trees surveyed at Burnaby Mountain but not concurrently isolated from adult beetles and gallery phloem samples from the same gallery. This putatively novel species of Ophiostoma was never isolated from larvae, pupae, or teneral adults. Cadophora spadicis and Penicillium spp. were the two most prominent filamentous fungal taxa that did not fall into Neonectria or ophiostomatoid fungus categories. Cadophora spadicis was the third most frequently isolated filamentous fungus species, yet it was never concurrently isolated from beetles and gallery phloem samples from the same gallery (Table 2). Penicillium spp., comprising up to 10 different species in our study, had an overall occurrence of 7.4-38.9% (Tables 1 and 2). All other observed filamentous fungus species were rarely isolated. Except Beauveria spp., which occurred on 5.6% of adult beetles, these fungi were each isolated from only one or two adult beetle or gallery phloem samples ( Table 2).
Ophiostoma quercus (Georgev.) Nannf. (Ophiostomatales: Ophiostomataceae) was the second most frequently isolated fungal associate of A. aspericollis, yet it was infrequently isolated from adult beetles and even more rarely cultured from gallery phloem. Ophiostoma quercus is a sapwood-staining ophiostomatoid fungus that occurs worldwide in a variety of hardwood genera (e.g., Quercus, Fagus, Acer, Betula, Eucalyptus, Castanea) [60] and is considered to be saprotrophic or mildly pathogenic [72,73]. Ophiostoma quercus has been found on Alnus incana in Latvia [74], but our study is the first to report O. quercus from A. rubra. As a casual associate of many hardwood-invading bark beetles worldwide [75,76], it was probable that O. quercus would be occasionally isolated from A. aspericollis. Our study also reports the discovery of a new ophiostomatoid species, Ophiostoma sp. nov., a minor associate of A. aspericollis. Ophiostoma sp. nov. was only found at one of seven sampling sites, Burnaby Mountain, where it was isolated slightly more frequently than O. quercus from all beetle-infested trees but only occasionally cultured from adult A. aspericollis and gallery phloem. Many bark beetle species have more than one ophiostomatoid fungal partner [76][77][78], and the relative dominance of different ophiostomatoid partners may vary with geographic location and environmental conditions [28,79].
Other tree pathogens and decay fungi, including V. oxystoma and C. spadicis, as well as various ubiquitous saprotrophs and the entomopathogenic Beauveria spp., were identified on A. aspericollis and in gallery phloem. These fungi were rarely isolated, however, and were obtained from either adult beetles or gallery phloem samples, but not both. For instance, Beauveria spp. were isolated from adult beetles only, which is consistent with the entomopathogenic function of these fungi [80,81]. Likewise, fungi such as V. oxystoma, for which A. rubra is a known host, and C. purpureum were only isolated from gallery phloem samples, which is consistent with their ecological roles as tree pathogens and decay fungi [82,83].
This study represents the first record of C. spadicis (Prodi, Sandalo, Tonti, Nipoti & A. Pisi) Travadon, Lawrence, Rooney-Latham, Gubler, Wilcox, Rolshausen & K. Baumgartner (Order: Helotiales) on red alder, and to the best of our knowledge, in western Canada. The genus Cadophora comprises various plant pathogens, decay fungi, and saprotrophs [50,53,84,85]. Cadophora spadicis has been reported from grapevine, Vitis spp., in North America [50], is known to induce trunk hypertrophy in kiwifruit, Actinia deliciosa, in Italy [50,53], and is associated with hardwood-infesting longhorn beetles (Coleoptera: Cerambycidae) in Finland [85]. Other Cadophora species have been occasionally isolated from Alnus incana and A. glutinosa [74,83]. Cadophora spadicis was infrequently isolated from A. aspericollis and gallery phloem samples and could be a decay or pathogenic fungus of red alder that occasionally utilizes the entrance holes of A. aspericollis for access to host trees, and may therefore be coincidentally carried by the beetles. This passive association is also the likely case for other generalist saprotrophs (e.g., Penicillium spp., Cladosporium sp.) that were isolated during this study. These ubiquitous wind-dispersed fungi have been found in similar frequencies in other bark beetle systems [77,86] and are not known to colonize living trees nor to be antagonists of bark beetles, unlike those reported by [87,88] (e.g., Trichoderma harzianum, T. polysporum, Aspergillus fumigatus, A. nomius). The ubiquitous saprophytic species identified by this study are therefore likely to be late colonizers of A. aspericollis-killed trees, or opportunistic hitchhikers of A. aspericollis, and are probably of little ecological importance to the beetle.
In keeping with our initial prediction, A. aspericollis did not display a consistent relationship with ophiostomatoid fungi but was instead casually associated with several species (i.e., O. quercus, Ophiostoma sp. nov., and G. fragrans). Conifer-killing bark beetles tend to be consistently associated with specific ophiostomatoid fungi [20,29], with the possible exception of a few resource-pulse driven species (e.g., Tomicus piniperda, Hylurgus ligniperda) [77,89]. Given that the host tree of A. aspericollis, A. rubra, fixes atmospheric nitrogen and its tissues are thereby rich in this limiting nutrient [37], selection for an association with ophiostomatoid fungi based upon their capacity to concentrate nitrogen in the phloem of their host trees [22,23] seems unlikely. The absence of an intimate association between A. aspericollis and ophiostomatoid fungi lends support to the assertion that the mutualistic relationship between conifer-killing bark beetles and ophiostomatoid fungi arose in part from the fungi's capacity to improve the nutritional condition of phloem for developing beetles [21][22][23]. The loose association between A. aspericollis and ophiostomatoid fungi strongly suggests that these fungi do not provide any significant ecological benefits to A. aspericollis.
Neonectria sp. nov. was frequently isolated from both adult beetles and phloem within the same gallery, suggestive of a fungus-vector relationship where A. aspericollis is capable of transmitting Neonectria sp. nov. between host red alder trees. It is unclear, however, if Neonectria sp. nov. has an obligate or facultative relationship with A. aspericollis since the fungus was not isolated at the high frequencies (i.e., 90-100%) observed in well-established bark beetlefungus symbioses (e.g., Dendroctonus rufipennis and Leptographium abietinum, Dendroctonus ponderosae and Ophiostoma spp., Scolytus scolytus and O. ulmi) [90][91][92]. Little is known about the etiology of N. major, but reproductive spores of the closely-related Neonectria ditissima (Tul. & C. Tul.) Samuels and Rossman are usually wind or water-dispersed, gaining entry to host trees via stem wounds, bark cracks, and leaf scars [93]. Cootsona [67] suggested that drought stress predisposes red alder to N. major infection in the Pacific Northwest and did not discount the possibility that insects could vector N. major. The pathogenicity of Neonectria sp. nov. remains untested, and we observed during sample collection that Neonectria sp. nov. infection in A. aspericollis-infested red alder was not accompanied by typical Neonectria disease symptoms. Canker pathogens including Neonectria and Geosmithia species usually induce target-shaped cankers or prominent lesions to form on the limbs or stems as infections progress [67,93,94]. None of the red alders sampled during this study displayed prominent canker or lesion symptoms on their main stems, raising the possibility that Neonectria sp. nov. may proliferate throughout tree tissues in a different manner when transmitted by A. aspericollis than when the fungus infects a host on its own.
Hardwood-infesting bark beetles maintain relationships of varying degrees of intimacy with both ophiostomatoid fungal associates and non-ophiostomatoid fungi including representatives from the order Hypocreales, the taxon that includes Neonectria and Geosmithia. Scolytus scolytus nearly always carries O. ulmi as part of the Dutch elm disease complex [90], while S. multistriatus and S. schevyrewi are associated with O. novo-ulmi at inconsistent frequencies (i.e., 9-92%) [95]. The birch bark beetle S. ratzeburgi is consistently associated with O. karelicum, a potentially pathogenic fungus that may be etiologically similar to Dutch elm disease [59,96]. Scolytus intricatus and Hylesinus varius, which kill their respective weakened oak and ash hosts, are loosely and infrequently associated with ophiostomatoid fungi, including the non-virulent O. quercus [75,97]. Pseudopityophthorus spp. are known to transmit oak wilt, B. fagacearum [32,98]. The walnut twig beetle, Pityophthorus juglandis, vectors the phytopathogen Geosmithia morbida, a beetle-fungus complex responsible for the thousand canker disease of black walnut in the western United States, yet in most cases Geosmithia spp. associates of bark beetles are not considered to be pathogenic [71,94]. Ceratocystis spp. thought to be vectored by the mango bark beetle, Hypocryphalus mangiferae, cause diseases of mango trees in plantations worldwide [99][100][101].
Future research should focus on clarifying the ecological relationship between A. aspericollis and its Neonectria sp. nov. partner, specifically whether this association is strictly casual or representative of a symbiosis that is mutualistic, commensalistic, or antagonistic in nature. To determine whether A. aspericollis is a vector of Neonectria sp. nov., experimental work should be directed toward addressing the criteria outlined by Leach [102] for defining a pathogenvector relationship. Further, assessing the effects of Neonectria sp. nov. on the host colonization and reproductive success of A. aspericollis would reveal whether this potential symbiosis represents a mutualism. The Neonectria sp. nov. isolates obtained during this study were nearly genetically identical, suggesting that this species originated from a single and recent evolutionary event that is worthy of future investigation. Finally, additional work is required to morphologically describe the novel species of Neonectria and Ophiostoma identified by phylogenetics herein.
Supporting information S1 Table. Strains of Neonectria sp. nov., Ophiostoma quercus, Ophiostoma sp. nov., and Cadophora spadicis isolated from Alniphagus aspericollis and gallery phloem, including their collection site(s) and GenBank accession numbers for barcode sequences (internal transcribed spacer (ITS) rDNA gene region, partial beta-tubulin (BT) gene, partial translation elongation factor 1-alpha (EF1-α) gene, and partial RNA polymerase II second largest subunit (RPB2) gene). Mycelia from each isolate comprising the listed strains, with the exception of several O. quercus isolates that were not successfully sub-cultured after initial investigation, were preserved in 10% glycerol, flash-frozen in liquid nitrogen, and placed at -80˚C for long-term storage in the Genomics and Forest Health (Hamelin) Lab at the University of British Columbia, Canada. (DOCX)